Today, about two-thirds of U.S. adults are overweight or obese, according to the Centers for Disease Control and Prevention. Obesity is harmful to physical health as well as an established risk factor for a number of potentially life-threatening diseases such as atherosclerosis, hypertension, diabetes, stroke, pulmonary embolism, and cancer. Moreover, obesity can wreak havoc on an individual's mental health and can affect a person's ability to interact socially with others.
Accompanying the devastating medical consequences of this problem is the severe financial burden placed on the health care system in the United States. The estimated economic cost of obesity and its associated illnesses from medical expenses and loss of income are reported to be in excess of $68 billion per year. Because of the impact of obesity on individuals and society, much effort has been expended to find ways to treat obesity, but little success has been achieved in the long-term treatment and/or prevention of obesity.
Pro-opiomelanocortin (POMC) derived peptides are known to affect food intake. Several lines of evidence support the notion that the G-protein coupled receptors (GPCRs) of the melanocortin receptor (MCR) family, several of which are expressed in the brain, are the targets of POMC derived peptides involved in the control of food intake and metabolism.
Five distinct MCRs have thus far been identified, and these are expressed in different tissues. MC1R was initially characterized by dominant gain of function mutations at the Extension locus, affecting coat color by controlling phaeomelanin to eumelanin conversion through control of tyrosinase. MC1R is mainly expressed in melanocytes. MC2R is expressed in the adrenal gland and represents the ACTH receptor. MC3R is expressed in the brain, gut, and placenta and may be involved in the control of food intake and thermogenesis. MC4R is uniquely expressed in the brain, and laboratory observations suggest that it is also involved in the control of food intake. See Kask A, et al., “Selective antagonist for the melanocortin-4 receptor (HS014) increases food intake in free-feeding rats,” Biochem. Biophys. Res. Commun., 245:90–93 (1998)). MC5R is expressed in many tissues, including white fat, placenta and exocrine glands. MC5R knockout mice reveal reduced sebaceous gland lipid production (Chen et al., “Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides,” Cell, 91:789–798 (1997)).
Evidence for the involvement of MCRs in obesity includes: a) the agouti (Avy) mouse which ectopically expresses an antagonist of the MC1R, MC3R and MC4R is obese, indicating that blocking the action of these three MCRs can lead to hyperphagia and metabolic disorders; b) MC4R knockout mice (Huszar, D. et al., “Targeted disruption of the melanocortin-4 receptor results in obesity in mice,” Cell, 88:131–141 (1997)) recapitulate the phenotype of the agouti mouse—these mice are obese; c) the cyclic heptapeptide MT-II (a non-selective MC1R, MC3R, MC4R, and MC5R agonist) injected intracerebroventricularly (ICV) in rodents, reduces food intake in several animal feeding models (NPY, ob/ob, agouti, fasted) while ICV injected SHU-9119 (MC3R and MC4R antagonist; MC1R and MC5R agonist) reverses this effect and can induce hyperphagia; iv) chronic intraperitoneal treatment of Zucker fatty rats with an NDP-MSH derivative (HP228) has been reported to activate MC1R, MC3R, MC4R, and MC5R and to attenuate food intake and body weight gain over a 12-week period (Corcos, I. et al., “HP228 is a potent agonist of melanocortin receptor-4 and significantly attenuates obesity and diabetes in Zucker fatty rats,” Society for Neuroscience abstracts, 23:673 (1997)).
A specific single MCR that may be targeted for the control of obesity has not yet been identified, although evidence has been presented that MC4R signaling is important in mediating feed behavior (Giraudo, S. Q. et al., “Feeding effects of hypothalamic injection of melanocortin-4 receptor ligands,” Brain Research, 80:302–306 (1998)) and MC3R signaling may decrease food intake and participate in the regulation of energy homeostasis (obesity).
Agouti-related protein (AGRP) is a 132 (human) amino acid peptide putatively containing five disulfide bridges, and antagonizes the central brain melanocortin receptors (MC3R and MC4R) (Ollmann, M. M. et al., “Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein,” Science, 278:135–138 (1997); and Yang, Y. K. et al., “Characterization of Agouti-related protein binding to melanocortin receptors,” Mol. Endo., 13:148–155 (1999)). Agouti (ASP) is a homologue of AGRP and was first identified as an endogenous G-protein coupled receptor (GPCR) antagonist. Both of these proteins are the only known naturally occurring antagonists of GPCRs reported to date, making them a unique family of peptides.
Previous structure-activity studies of the agouti peptide identified the importance of the three amino acid motif Arg-Phe-Phe that is conserved in both agouti and AGRP (see, for example, Kiefer, L. et al., “Mutations in the carboxyl terminus of the agouti protein decrease agouti inhibition of ligand binding to the melanocortin receptors,” Biochemistry, 36:2084–90 (1997)). These studies suggest that the conserved Arg-Phe-Phe motif found in both agouti and AGRP may be important for the antagonistic and molecular recognition properties of these two molecules at the melanocortin receptors.
All endogenous melanocortin agonists contain the putative amino acid sequence (His)/Phe-Arg-Trp, postulated to be important for melanocortin receptor molecular recognition and stimulation. Further extrapolation of the homology between the antagonist Arg-Phe-Phe motif and the endogenous melanocortin agonist conserved residues Phe-Arg-Trp, implies that the antagonist residues may be mimicking the agonist Phe-Arg-Trp interactions with the melanocortin receptors, as supported by Tota, M. R., et al., “Molecular interaction of Agouti protein and Agouti-related protein with human melanocortin receptors,” Biochemistry, 38:897–904 (1999) and Haskell-Luevano, C., et al., “The agouti-related protein decapeptide (Yc[CRFFNAFC]Y) possesses agonist activity at the murine melanocortin-1 receptor,” Peptides, 21:683–689 (2000).
Fragments of the agouti protein have been reported to be MC1R agonists (Yang, Y. K., et al., “Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand,” Am. J. Physiol. Regul. Integr. Comp. Physiol., 281:R1877–1886 (2001)). Various melanocortin agonist peptides (i.e., Ac-His-DPhe-Arg-Trp-NH2 and Ac-His-Phe-Arg-Trp-NH2) have been reported to possess nM and μM potencies, respectively, at the mouse melanocortin receptors, and that the tripeptide Ac-Phe-Arg-Trp-NH2 possesses μM agonist activity at the mMC1R (Haskell-Luevano, C., et al., “Characterization of melanocortin NDP-MSH agonist peptide fragments at the mouse central and peripheral melanocortin receptors,” J. Med. Chem, 44:2247–2252 (2001)). Further studies have shown that the Ac-His-Phe-Arg-Trp-NH2 is the minimal fragment of melanocortin agonists required to produce a physiological response (μM) in the classic frog and lizard skin bioassay Hruby, V. J., et al., “alpha-Melanotropin: the minimal active sequence in the frog skin bioassay,” J. Med. Chem., 30:2126–2130 (1987); and Castrucci, A. M. L., et al., “Alpha-melanotropin: the minimal active sequence in the lizard skin bioassay,” Gen. Comp. Endocrinol, 73:157–163 (1989).
In view of the need to better understand the biology of obesity and its relationship with MCRs, novel agents, methods, and compositions for treating or preventing obesity need to be identified and developed.